Alice:LabNotes/2008-11-4: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Zsakura2
 
(4 intermediate revisions by 2 users not shown)
Line 8: Line 8:
  Add the following reagents along with the spacers then denature at 95C for 5 min, anneal at 65c for 15 min.  
  Add the following reagents along with the spacers then denature at 95C for 5 min, anneal at 65c for 15 min.  
   
   
  template (1) 680nM                 1.5 ul  
  template (1) 680nM (actually 211nM) 1.5 ul  
  spacer mix (100nM each)              20 ul
  spacer mix (100nM each)              20 ul
  10x Ampligase Buffer                  5 ul
  10x Ampligase Buffer                  5 ul
Line 34: Line 34:
  TBE gel result:
  TBE gel result:
  [[Image:ZhangLab_2 2008-11-06 16hr 17min.jpg]]
  [[Image:ZhangLab_2 2008-11-06 16hr 17min.jpg]]
1: PCR product of cosmic probe
2: PCR product after spacer insertion
Conclusion: The TBE gel result of the PCR product after spacer insertion showed two clear bands, one having the same size as lane 1,
and the other band having extra 60bp in length. This shows that the spacer is successfully inserted into the template.

Latest revision as of 22:50, 9 January 2009

diagnosing spacer annealing and insertion efficiency[edit]

For the product and procedure from previous steps, please follow this link (10/14/08 LabNote):  [[1]]
Add 1ul of spacer to 99ul of ddH2O to dilute them to 100nM
the final concentration of spacer is 20nM, so 20nM x 50ul =  100nM x amount needed(ul)           
Add 10ul of each primer, denature at 95C for 5min, and then anneal at 65C for 15 min.

Add the following reagents along with the spacers then denature at 95C for 5 min, anneal at 65c for 15 min. 

template (1) 680nM (actually 211nM) 1.5 ul 
spacer mix (100nM each)              20 ul
10x Ampligase Buffer                  5 ul
Ampligase(5U/ul)                      1 ul
ddH2O                              15.6 ul
------------------------------------------
total                                50 ul

Add 1ul of dNTP, 0.5ul of Stoffel fragment to the reaction system above, 60C for 20min. <----unnecessary
File:ZhangLab 2 2008-11-04 16hr 33min.jpg
TBE gel result:
1: product from reaction system above(after spacer insertion)
2: cosmic probe product from PCR

Add 2ul of ExoI&III 37C for 2hrs -> 94C 10min

PCR amplification after spacer insertion (11/6/08)[edit]

H2O                                  40ul
SYBG                                0.4ul
primer mix (AP1V4IU+AP2V4)          0.2ul
Taq master mix                       50ul
Template (from previous step)        10ul
----------------------------------------
total                               100ul
TBE gel result:
File:ZhangLab 2 2008-11-06 16hr 17min.jpg
1: PCR product of cosmic probe 
2: PCR product after spacer insertion

Conclusion: The TBE gel result of the PCR product after spacer insertion showed two clear bands, one having the same size as lane 1,
and the other band having extra 60bp in length. This shows that the spacer is successfully inserted into the template.